ABSTRACT/SUMMARY Despite suppression by antiretroviral therapy (ART), virus is not eliminated in HIV patients and can rebound causing full-blown infection upon ART interruption. Thus, a strategy to eliminate the virus reservoir is urgently needed. The recently discovered gene editing technique called CRISPR has tremendous potential for eradicating HIV-1. CRISPR is comprised of a Cas9 nuclease and chimeric guide RNA (gRNA). When Cas9 and gRNA designed to target HIV sequences are present in latently-infected cells, it can result in disruption of the integrated proviral genome, permanently inactivating the virus. The biggest challenge to using the CRISPR approach for HIV elimination is the absence of an in vivo delivery system for human T cells, the major cellular of HIV-1. This is a R01 application in response to RFA-AI-18-016, ?Targeted In Vivo Delivery of Gene Therapeutics for HIV Cure?. To address the challenge of Cas9/gRNA delivery to human T cells in vivo, we propose the use of a T cell-targeting lentivirus whose tropism is guided by antibodies to human CD7, a molecule expressed at high levels on all human T cells, including resting T cells which are a major reservoir for latent HIV. To address concerns of vector integration and constitutive Cas9 expression, we have generated lentiviruses that are pre-packaged with Cas9 ribonucleoproteins with no integrating DNA components. Proof- of-concept studies in virologically-suppressed HIV-infected humanized mice demonstrate that disrupting CCR5, the coreceptor for HIV-1, with this systemic approach results in ART-free virologic remission. Importantly, as the approach does not require activation or elimination of the infected cell, it addresses the limitations of conventional `Shock and Kill' approaches that have yielded promising results in clinical settings. The proposal has three specific aims- In Specific Aim 1, we will design and test broad-spectrum gRNAs targeting HIV DNA in two independent approaches expected to mutate a segment or excise the entire length of the integrated HIV provirus. The approach will be tested in ART-suppressed humanized mice for impact on virus reservoir and rebound. A comprehensive investigation of toxicity, off-target effects and virus escape will also be undertaken. In Specific Aim 2, we will perform functional studies in patient-cell derived humanized mice to determine the effects of broad-spectrum gRNAs on latent virus quasispecies from HIV+ patients. The studies will employ HIV-1 RNA Sort-Seq, a novel methodology to quantitate the inducible replication-competent HIV reservoir. In Specific Aim 3, we incorporate strategic changes in the lentiviral vector to reduce vector-associated immunogenicity and permit a single or multiple but rapid-dosing regimen with enhanced potency. The outcome of these proof-of-principle studies is expected to establish a solid platform for future studies on an approach that could significantly contribute towards a cure for HIV-AIDS.